Cling-E. coli : Bacteria on target Harvard iGEM 2007 Ellenor Brown Stephanie Lo Alex Pickett Sammy Sambu Kevin Shee Perry Tsai Shaunak Vankudre George Xu

Harvard iGEM 2007 Introduction The motivation To develop a system for directing bacteria to a target of interest and effecting downstream activity

Harvard iGEM 2007 Introduction Bind Proteins Bind Other Cells Bind Tissue Bind Surface Bind DNA Bind Viruses Bind Toxins Potential Targets and Applications

Harvard iGEM 2007 Introduction Quorum-sensingFec signal transduction Bacterial targeting Quorum-sensingFec signal transduction

Harvard iGEM 2007 Bacterial Targeting Surface Engineered Bacteria Engineered to Bind and Signal Fusion Protein Membrane Protein OmpA – C terminal insertion OmpA-Loop1 insertion AIDA-1 – N terminal insertion

Harvard iGEM 2007 Bacterial Targeting Selecting/enriching for surface engineered bacteria Magnetism Activated Cell Sorting (MACS) Fluorescence Activated Cell Sorting (FACS) – His tag + nickel beads – Strep2 tag + streptavidin beads Fluorescence Bead Assays Magnetic Bead Assays

Harvard iGEM 2007 Bacterial Targeting His/Strep2 –tagged bacteria are enriched by MACS Magnetic Bead Assays

Harvard iGEM 2007 Bacterial Targeting Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) - Assay Results his strep2 Before Separation After Separation

Harvard iGEM 2007 Bacterial Targeting Results: Cell Selection Assays are a Success! Selecting for surface engineered bacteria (such as his and strep2) is possible through either direct or indirect cell separation.

Harvard iGEM 2007 Quorum Sensing Bacterial targeting Fec signal transductionQuorum-sensing

Harvard iGEM 2007 Quorum Sensing luxI/luxR Quorum Sensing Sender + R OHHL Receiver

Harvard iGEM 2007 Quorum Sensing Receivers (luxR + Reporter) –GFP Receivers tetR controlled (Bba_T9002) Quorum controlled (Bba_R Bba_C Bba_E0240) –mRFP Receivers tetR controlled (Bba_F Bba_I13507) Quorum controlled (Bba_R Bba_C Bba_I13507) –mCherry Receivers (Bba_F Bba_J06702) Senders (bicistronic luxI + Reporter) –mRFP Sender tetR controlled (Bba_S Bba_I13507) lacI controlled (Bba_S Bba_I13507) Quorum controlled (Bba_R Bba_A Bba_I13507) –GFP Sender tetR controlled (Bba_S Bba_E0240) lacI controlled (Bba_S Bba_E0240) Quorum controlled (Bba_R Bba_A Bba_E0240) –mCherry Sender tetR controlled (Bba_S Bba_J06702) Single Cell –Constitutive (Bba_J Bba_T9002) –Quorum Controlled (Bba_R Bba_A Bba_C Bba_E0240) Construction Intermediates Cell-Cell Signaling Constructs Receiver Sender

Harvard iGEM 2007 Quorum Sensing Switch-like Quorum Response Sender Receiver

Harvard iGEM 2007 Quorum Sensing Selection with Direct Magnetic Beads Control: no selection Experimental: Selection with beads

Harvard iGEM 2007 Quorum Sensing 60-fold Enrichment through Direct Magnetic Beads Control: no beadsSelection with streptactin beads

Harvard iGEM 2007 Quorum Sensing Enriched senders activate quorum response Receiver Sender

Harvard iGEM 2007 Fec signal transduction Bacterial targeting Quorum-sensingFec signal transduction

Harvard iGEM 2007 Fec signal transduction Motivation: Fec System Goal: Direct cell signaling Method: Re-engineer an existing signal transduction pathway Fec system: – well-characterized – substrate specific

Harvard iGEM 2007 Fec signal transduction Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2): Ferric citrate

Harvard iGEM 2007 Fec signal transduction Overview of Fec System Ferric citrate Loops 7 & 8

Harvard iGEM 2007 Fec signal transduction Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2): Ferric citrate

Harvard iGEM 2007 Fec signal transduction Fec: Motivation Re-engineer FecA: Mutate loop 7 and/or loop 8 Structural Evidence: –L7 moves up to 11Å, helix unwinds –L8 moves up to 15Å Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) Loops 7 & 8

Harvard iGEM 2007 Fec signal transduction Constructs From Braun lab (Germany) –Fec knock-out strain, AA93 –FecIRA plasmid –Fec promoter, GFP plasmid pCola Duet Vector –Allows regulated expression of Fec genes under T7 promoter

Harvard iGEM 2007 Fec signal transduction Wild-Type GFP Expression

Harvard iGEM 2007 Fec signal transduction Troubleshooting and Next Steps Problems: –Growth media –Toxicity: membrane disruption? Goals: –Nickel and Streptavidin Binding –Finding new targets with signaling Random library Computational Approach

Harvard iGEM 2007 Conclusion Conclusions Targeting – His and Strep2 tags on AIDA, targeting bacteria to nickel and streptavidin was successful Quorum sensing – Constructed one-cell system – Characterized two-cell system – Combined with targeting Fec signal transduction –Characterized Fec system

Harvard iGEM 2007 Conclusion Future Directions Bacterial targeting –Trying out new targeting peptides (calmodulin) –Optimizing the random library approach in selecting for targeting peptides Quorum sensing –Characterizing one-cell system –Optimizing quorum response after targeting Fec signal transduction –Effect signal transduction with targeting Application

Harvard iGEM 2007 Conclusion Acknowledgements Advisors George Church Debra Auguste Jagesh V. Shah William Shih Pamela Silver Alain Viel Tamara Brenner Teaching Fellows Nicholas Guido Bill Senapedis Mike Strong Harris Wang Funding HHMI Harvard Provost Harvard Life Sciences Division Harvard School of Engineering and Applied Sciences

Harvard iGEM 2007 Bacterial Targeting N terminus modification of AIDA1

Harvard iGEM 2007 Bacterial Targeting C terminus modification of OmpA Post-Assay Plates Pre-assay plates

Harvard iGEM 2007 Bacterial Targeting CSR by gene Design Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) – PCR product insertion (950 bps) – Insertion of ds oligos

Harvard iGEM 2007 Bacterial Targeting CSR by gene design

Harvard iGEM 2007 Bacterial Targeting Bacterial Targeting: Cell Surface Reengineering (CSR) CSR by PCR product digestion & ligation: – Fusion of peptides to the C terminus of OmpA – Fusion of peptides to the N terminus of AIDA1 Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) – PCR product insertion (950 bps) – Insertion of ds oligos